Fig. 2. An antibody specific for Bpag1 isoform 2 detects protein co-aligning with actin stress fibers. (A) Western blot analysis of lysates from Cos-1 cells expressing Bpag1 fusion proteins. GFP-Nterm1 and GFP–Nterm2 were expressed, as shown with an anti-GFP antibody, and used to test anti-Bpag1 sera. Serum containing antibodies against the Bpag1 A' region detected GFP-Nterm2 but not GFPNterm1. GFP-Nterm2 was not detected with the pre-immune serum (data not shown), and was strongly detected with the affinity-purified antibody (A' Ab). (B) Only diffuse background staining was ever observed in cells stained with the pre-absorbed A' antibody. (C,D) In C2C12 cells, the A' antibody (C) stained in a filamentous pattern co-aligning with centrally located actin stress fibers (stained with rhodamine-phalloidin in D; closed arrows in C and D). The A' staining typically did not occur along more peripheral actin filaments (open arrows in C and D), although colocalization did occur along more peripheral actin filaments in a few cells. Strong peri-nuclear staining was also observed in a few cells (arrowhead in C). Bars, 20 µm.